Chemiluminescent Western Blot Detection

As with other components in a western blotting system, there are many chemiluminescent western blotting substrate choices available. The appropriate selection of a substrate depends on the detection level (sensitivity) required, the target protein's abundance, the sample abundance and antibody availability. Use the tables below to find the right horseradish peroxidase (HRP) or alkaline phosphatase (AP) substrate for your experimental needs.

Find the right chemiluminescent substrate

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SuperSignal West Femto

SuperSignal West Femto
Most sensitive
Sensitivity: Low femtogram
Duration: 8 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:5,000
2°: 1:100,000-1:500,000
When to use: Target is least abundant, sample is precious, maximum sensitivity

SuperSignal West Pico Plus

SuperSignal West Pico Plus
Wide dynamic range
Sensitivity: Low picogram to femtogram
Duration: up to 24 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:20,000-1:100,000
When to use: Everyday applications, improved sensitivity over base level ECL

SuperSignal West Dura

SuperSignal West Dura
Longest duration
Sensitivity: mid femtogram
Duration: up to 24 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:5,000
2°: 1:50,000-1:250,000
When to use: Target is less abundant, maximum signal duration is needed

Pierce ECL

Pierce ECL
Base level detection
Sensitivity: picogram
Duration: up to 2 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:1,000-1:15,000
When to use: Target and sample are abundant, lower sensitivity is needed

Pierce ECL Plus

Pierce ECL Plus
Chemi-fluorescent detection
Sensitivity: low picogram
Duration: up to 5 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:25,000-1:200,000
When to use: Flexibility in detection modes-Chemiluminescent and fluorescent properties

Additional products for HRP substrates

Thermo Fisher Scientific product Description Order
SuperSignal Western Blot Enhancer Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it. 46641
46640

Novex AP Chemiluminescent Substrate

Novex AP Chemiluminescent Substrate
Long signal duration
Sensitivity: low picogram
Duration: over 24 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:500-5,000
2°: 1:2,000-1:10,000
When to use: Used standalone substrate for PVDF membranes Add Nitro Block II (sold separately) to substrate for use with nitrocellulose membranes

Additional products for AP substrates

Thermo Fisher Scientific product Description Order
Novex AP Chemiluminescent Substrate Enhancer (Nitro Block II) 20X solution of Nitro-Block-II intended for use in conjunction with Novex AP Chemiluminescent Substrate to increase the intensity of chemiluminescent signals when probing blots on nitrocellulose membranes WP20003
WesternBreeze Chromogenic Kit, anti-rabbit Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodies WB7104
WesternBreezeChemiluminescent Kit, anti-rabbit Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodies WB7106
WesternBreeze Chemiluminescent Kit, anti-goat Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodies WB7108

Chemiluminescent detection

Chemiluminescent detection occurs when energy from a chemical reaction is released in the form of light. The two most common enzyme reporters that catalyze chemiluminescent reactions required for generating a recordable signal are horseradish peroxidase (HRP) and alkaline phosphatase (AP).

  HRP substrates (Horseradish peroxidase) AP substrates (Alkaline phosphatase)
Sensitivity

Femogram sensitivity

Picogram sensitivity
Signal generation Immediate Gradually increases with signal maximum at ~30-60 minutes
Signal duration Up to 24 h 24-96 hours
Considerations Compatible with common buffers such as TBS and PBS Not compatible with phosphate buffers
When to use Antibodies or Probes conjugated to HRP Antibodies or Probes conjugated to AP

Horseradish peroxidase (HRP) chemiluminescent substrates

HRP (40 kDa) has become the standard enzyme for chemiluminescent western blot detection. Its popularity grew due to its stability and smaller size, which enables more molecules conjugated per IgG providing greater sensitivity. Furthermore, advancement and improvements in chemiluminescent substrates for HRP has enabled even higher sensitivity over AP substrates for western blotting.

Majority of chemiluminescent substrates for horseradish peroxidase (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.

Although x-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, cooled CCD cameras offer the advantages of qualitative analysis, instant image manipulation, higher sensitivity, greater resolution and a larger dynamic range than film.

We offer five types of chemiluminescent substrates for western blot detection with horseradish peroxidase enzyme (HRP). Use the table in the HRP tab [SJ1] to select the most appropriate HRP Chemiluminescent substrate based on abundance of your target protein of interest, abundance of sample containing the target protein, and the level of sensitivity and type of instrumentation available for detection.

Don’t know which substrate is right for you? Use the chemiluminescent substrates selection guide to choose the right substrate for your research needs.

Learn more about each SuperSignal substrate

Alkaline phosphatase (AP) chemiluminescent substrates

For western blot detection based on alkaline phosphatase (AP), 86 kDa, we offer our CDP-Star substrate that is designed to deliver picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.  The emitted light is stable up to 24-96 hours allowing for multiple exposures.

Choose from a standalone substrate, Novex AP Chemiluminescent Substrate or the WesternBreeze Chemiluminescent Immunodetection kits which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane. Novex AP Chemiluminescent Substrate should be combined with Nitro Block II for use with nitrocellulose membranes.

Light Emission mechanism for CDP-Star Substrate

Light Emission mechanism for CDP-Star Substrate.

Nitrocellulose membranes provide an inefficient environment for chemiluminescence from 1,2-dioxetane substrates, resulting in very low signal intensity. Nitro-Block-II membrane enhancer increases signal intensity on both nitrocellulose and PVDF membranes. Nitro-Block-II enhancers generate a hydrophobic environment on the membrane surface that increases the intensity of chemiluminescence. The effects of NitroBlock enhancer treatment on nitrocellulose and PVDF membranes are shown. Without Nitro-Block enhancer, the signal on nitrocellulose membranes is weak, making detection either impossible or requiring extremely long exposure times. With Nitro-Block enhancer, short exposures of 10 to 45 minutes are achieved. While PVDF membranes does not require the use of Nitro-Block enhancer, exposure times are tenfold faster with Nitro-Block enhancer. This permits very short exposure times on PVDF, ranging from 15 seconds to 15 minutes for Western blots.

Chemiluminescent signal enhancement on Nitrocellulose and PVDF membranes with Nitro-Block Enhancer using a short exposure time
Chemiluminescent signal enhancement on Nitrocellulose and PVDF membranes with Nitro-Block Enhancer using a short exposure time.

SuperSignal West Femto

SuperSignal West Femto
Most sensitive
Sensitivity: Low femtogram
Duration: 8 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:5,000
2°: 1:100,000-1:500,000
When to use: Target is least abundant, sample is precious, maximum sensitivity

SuperSignal West Pico Plus

SuperSignal West Pico Plus
Wide dynamic range
Sensitivity: Low picogram to femtogram
Duration: up to 24 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:20,000-1:100,000
When to use: Everyday applications, improved sensitivity over base level ECL

SuperSignal West Dura

SuperSignal West Dura
Longest duration
Sensitivity: mid femtogram
Duration: up to 24 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:5,000
2°: 1:50,000-1:250,000
When to use: Target is less abundant, maximum signal duration is needed

Pierce ECL

Pierce ECL
Base level detection
Sensitivity: picogram
Duration: up to 2 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:1,000-1:15,000
When to use: Target and sample are abundant, lower sensitivity is needed

Pierce ECL Plus

Pierce ECL Plus
Chemi-fluorescent detection
Sensitivity: low picogram
Duration: up to 5 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:1,000
2°: 1:25,000-1:200,000
When to use: Flexibility in detection modes-Chemiluminescent and fluorescent properties

Additional products for HRP substrates

Thermo Fisher Scientific product Description Order
SuperSignal Western Blot Enhancer Membrane treatment reagent and a primary antibody diluent that increases both signal intensity and sensitivity 3- to 10-fold compared to detection performed without it. 46641
46640

Novex AP Chemiluminescent Substrate

Novex AP Chemiluminescent Substrate
Long signal duration
Sensitivity: low picogram
Duration: over 24 hours
Recommend antibody dilutions (1 mg/ml stock):
1°: 1:500-5,000
2°: 1:2,000-1:10,000
When to use: Used standalone substrate for PVDF membranes Add Nitro Block II (sold separately) to substrate for use with nitrocellulose membranes

Additional products for AP substrates

Thermo Fisher Scientific product Description Order
Novex AP Chemiluminescent Substrate Enhancer (Nitro Block II) 20X solution of Nitro-Block-II intended for use in conjunction with Novex AP Chemiluminescent Substrate to increase the intensity of chemiluminescent signals when probing blots on nitrocellulose membranes WP20003
WesternBreeze Chromogenic Kit, anti-rabbit Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using mouse primary antibodies WB7104
WesternBreezeChemiluminescent Kit, anti-rabbit Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using rabbit primary antibodies WB7106
WesternBreeze Chemiluminescent Kit, anti-goat Complete kit for detection of proteins transferred to nitrocellulose or PVDF membranes using goat primary antibodies WB7108

Chemiluminescent detection

Chemiluminescent detection occurs when energy from a chemical reaction is released in the form of light. The two most common enzyme reporters that catalyze chemiluminescent reactions required for generating a recordable signal are horseradish peroxidase (HRP) and alkaline phosphatase (AP).

  HRP substrates (Horseradish peroxidase) AP substrates (Alkaline phosphatase)
Sensitivity

Femogram sensitivity

Picogram sensitivity
Signal generation Immediate Gradually increases with signal maximum at ~30-60 minutes
Signal duration Up to 24 h 24-96 hours
Considerations Compatible with common buffers such as TBS and PBS Not compatible with phosphate buffers
When to use Antibodies or Probes conjugated to HRP Antibodies or Probes conjugated to AP

Horseradish peroxidase (HRP) chemiluminescent substrates

HRP (40 kDa) has become the standard enzyme for chemiluminescent western blot detection. Its popularity grew due to its stability and smaller size, which enables more molecules conjugated per IgG providing greater sensitivity. Furthermore, advancement and improvements in chemiluminescent substrates for HRP has enabled even higher sensitivity over AP substrates for western blotting.

Majority of chemiluminescent substrates for horseradish peroxidase (HRP) are two-component systems consisting of a stable peroxide solution and an enhanced luminol solution. To make a working solution, the two components are mixed together. When incubated with a blot on which HRP-conjugated antibodies (or other probes) are bound, a chemical reaction emits light at 425 nm which can be captured with x-ray film, CCD camera imaging devices and phosphorimagers that detect chemiluminescence. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.

Although x-ray film provides qualitative and semi-quantitative data and is useful to confirm the presence of target proteins, cooled CCD cameras offer the advantages of qualitative analysis, instant image manipulation, higher sensitivity, greater resolution and a larger dynamic range than film.

We offer five types of chemiluminescent substrates for western blot detection with horseradish peroxidase enzyme (HRP). Use the table in the HRP tab [SJ1] to select the most appropriate HRP Chemiluminescent substrate based on abundance of your target protein of interest, abundance of sample containing the target protein, and the level of sensitivity and type of instrumentation available for detection.

Don’t know which substrate is right for you? Use the chemiluminescent substrates selection guide to choose the right substrate for your research needs.

Learn more about each SuperSignal substrate

Alkaline phosphatase (AP) chemiluminescent substrates

For western blot detection based on alkaline phosphatase (AP), 86 kDa, we offer our CDP-Star substrate that is designed to deliver picogram level sensitivity and is compatible with both traditional x-ray film and CCD-based imaging. CDP-Star is dephosphorylated by AP to yield meta-stable dioxetane phenolate anion intermediate that decomposes and emits light with a maximum intensity at a wavelength of 475 nm. Light emission occurs only during the enzyme-substrate reaction; therefore, once the substrate in proximity to the enzyme is exhausted, signal output ceases.  The emitted light is stable up to 24-96 hours allowing for multiple exposures.

Choose from a standalone substrate, Novex AP Chemiluminescent Substrate or the WesternBreeze Chemiluminescent Immunodetection kits which contains all solutions necessary for your application including blocking solutions, primary antibody diluent, ready-to-use secondary antibody solution, ready-to-use chemiluminescent substrate, wash solutions, incubation trays, pre-cut filter papers, polyester sheet for even substrate development on the membrane. Novex AP Chemiluminescent Substrate should be combined with Nitro Block II for use with nitrocellulose membranes.

Light Emission mechanism for CDP-Star Substrate

Light Emission mechanism for CDP-Star Substrate.

Nitrocellulose membranes provide an inefficient environment for chemiluminescence from 1,2-dioxetane substrates, resulting in very low signal intensity. Nitro-Block-II membrane enhancer increases signal intensity on both nitrocellulose and PVDF membranes. Nitro-Block-II enhancers generate a hydrophobic environment on the membrane surface that increases the intensity of chemiluminescence. The effects of NitroBlock enhancer treatment on nitrocellulose and PVDF membranes are shown. Without Nitro-Block enhancer, the signal on nitrocellulose membranes is weak, making detection either impossible or requiring extremely long exposure times. With Nitro-Block enhancer, short exposures of 10 to 45 minutes are achieved. While PVDF membranes does not require the use of Nitro-Block enhancer, exposure times are tenfold faster with Nitro-Block enhancer. This permits very short exposure times on PVDF, ranging from 15 seconds to 15 minutes for Western blots.

Chemiluminescent signal enhancement on Nitrocellulose and PVDF membranes with Nitro-Block Enhancer using a short exposure time
Chemiluminescent signal enhancement on Nitrocellulose and PVDF membranes with Nitro-Block Enhancer using a short exposure time.